The study of modified nucleoside contributions to RNA chemistry, structure and function has been thwarted by the lack of a site-selected method of incorporation which is both versatile and adaptable to present synthetic technologies. A reproducible and versatile site-selected incorporation of nine differently modified nucleosides into hepta-and octadecamer RNAs has been achieved with automated phosphoramidite chemistry. A modified nucleoside-containing tRNA domain has been synthesized and purified in mmole quantities required for biophysical, as well as biochemical, studies. The anticodon domain of yeast tRNAPhe was synthesized with modified nucleosides introduced at the native positions: Cm32, Gm34, and m5C40. Spectroscopy at 750 MHz 1H will contribute significantly to the first 3D structure determination of a biologically functioning, modified RNA hairpin. The structure determination will provide evidence of modified nucleoside function in anticodon participation in translation.